Cultivated crops such as maize, soybean, and cotton have substantial commercial value throughout the world. The development of scientific methods useful in improving the quantity and quality of important crops is, therefore, of significant commercial interest. Significant effort has been expended to improve the quality of cultivated crop species by conventional plant breeding. Conventional means for crop and horticultural improvements utilize selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are often labor intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from the parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA) and the subsequent introduction of that genetic material into a plant's genome. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
The introduction of the foreign genetic material into a plant's genome is typically performed through one of two ways, although other ways are known to those skilled in the art. The first is biolistic particle bombardment, whereby the foreign DNA, or “transgene,” is coated onto a metal particle, which is then shot into plant tissue. Some of that foreign genetic material is taken up by the plant cells, which are thereby “transformed.” The second method is by Agrobacterium-mediated transformation, which involves exposing plant cells and tissues to a suspension of Agrobacterium cells that contain certain DNA plasmids. In both methods, the foreign DNA typically encodes for a selectable marker that permits transformed plant cells to grow in the presence of a selection agent, for example an antibiotic or herbicide. These cells can be further manipulated to regenerate into whole fertile transgenic plants.
For both Agrobacterium-mediated transformation and biolistic transformation, there is a possibility of the insertion of more than one copy of the transgene in a plant genome. For biolistic transformation, it is known in the art that a significant percentage of transgenic events contain more than one copy of the transgene. For Agrobacterium-mediated transformation, the transformation method utilizes a T-DNA (transferred DNA) that incorporates the genetic elements of a transgene and transfers those genetic elements into the genome of a plant. The transgene(s) are constructed in a DNA plasmid vector and are usually bordered by an Agrobacterium Ti plasmid right border DNA region (RB) and a left border DNA region (LB). During the process of Agrobacterium mediated transformation, the DNA plasmid is nicked by VirD2 endonuclease at the right and left border regions and the T-DNA region is inserted into the plant genome. The integration of the T-DNA into the plant genome generally begins at the RB and continues to the end of the T-DNA, at the LB. However, it has been found that more than one copy of the T-DNA is frequently inserted into the plant genome. (Tzfira et al 2004, Trends in Genetics, 20: 375-383; Windels et al, 2008, “Agrobacterium tumefaciens-mediated transformation: patterns of T-DNA integration into the host genome.” In Agrobacterium: from Biology to Biotechnology, Tzfira, T. and Citovsky, V., Eds. Springer, New York, N.Y.: 441-481, and references within).
It is important to produce transformed plants that have only one copy of the foreign DNA integrated into its genome. Significant resources are directed toward screening transformed plants for the presence of a single copy of the transgene. Methods such as TAQMAN™ analysis and Southern blot analysis are most frequently used. Typically, the initially transformed tissue has gone through several stages of selection and regeneration before it has enough tissue to be sampled for transgene copy number to be accurately ascertained by TAQMAN.™ Designing constructs to increase the incidence of a single insertion in the genome would be very valuable.